Methods for enriching for methylated nucleic acid molecules

Abstract

Disclosed are methods for enriching for nucleic acid molecules (e.g., enriching for methylated nucleic acid molecules), thereby enriching for target sequences of interest in a sample (e.g., a sample obtained from a subject). Methods disclosed herein involve using specifically designed methylation specific primers to generate single-stranded circular nucleic acids. The single-stranded circular nucleic acids are further enriched and analyzed to detect the target sequence.

Description

Attorney Docket No. HRG-030WOMETHODS FOR ENRICHING FOR METHYLATED NUCLEIC ACID MOLECULESCROSS REFERENCE

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 720,536, filed November 14, 2024, and U.S. Provisional Patent Application No. 63 / 766,851, filed March 4, 2025, the disclosure of each of which is hereby incorporated by reference in its entirety for all purposes.BACKGROUND

[0002] Non-invasive liquid biopsy tests are proving to be the next frontier in cancer diagnostics. There is a rising interest in sensitive, low-cost liquid biopsy assays to detect cancer-specific methylation events. Real-time quantitative methylation-specific PCR (qMSP) allows for the detection of rare methylated fragments, however widespread application has not been achieved. While qMSP achieves high specificity and sensitivity with low cfDNA inputs, assay design is challenged by the mostly three-base genome of bisulfite-converted DNA and the high homology between methylated ctDNA and excessive unmethylated cfDNA background.SUMMARY OF THE INVENTION

[0003] The disclosure relates to methods of detecting a target sequence in a nucleic acid sample. The target sequence may be indicative of the risk of developing or the presence of cancer in the subject from whom the sample was taken. In various embodiments, methods disclosed herein involve using specifically designed methylation specific primers to generate single-stranded circular nucleic acids. The single-stranded circular nucleic acids can be further enriched and analyzed to detect the target sequence. In various embodiments, the methods disclosed herein improve detection of nucleic acids containing a target sequence, e.g., rare target sequences, by isolating and / or enriching such target sequences in a nucleic acid sample. For example, the rare target sequence may be in a nucleic acid sequence from a cfDNA sample, such as a cfDNA sample that has been treated with bisulfite or chemical / enzymatic conversion to convert cytosines to uracils to preserve information regarding the methylation status of a particular nucleic acid sequence (e.g., comprising a CpG site), in a subject.Attorney Docket No. HRG-030WO

[0004] In one aspect, provided herein are methods for detecting a target sequence, the method comprising: obtaining converted nucleic acid molecules derived from a sample; providing one or more methylation specific primers to the converted nucleic acid molecules to generate one or more complexes comprising a methylation specific primer and a converted nucleic acid molecule, the methylation specific primer comprising a non-complementary region and flanking arms that flank the non-complementary region, at least one flanking arm hybridised to a target sequence of the converted nucleic acid molecule; generating a plurality of single -stranded circular nucleic acids using at least the methylation specific primers; enriching for the plurality of single-stranded circular nucleic acids; and detecting the target sequence from the plurality of single-stranded circular nucleic acids.

[0005] In some embodiments, the non-complementary region of the methylation specific primer comprises a rolling amplification sequence.

[0006] In some embodiments, the non-complementary region of the methylation specific primer comprises a restriction enzyme sequence.

[0007] In some embodiments, the non-complementary region of the methylation specific primer comprises a barcode sequence.

[0008] In some embodiments, the non-complementary region of the methylation specific primer comprises a biotinylated nucleotide sequence.

[0009] In some embodiments, the non-complementary region of the methylation specific primer comprises a rolling amplification sequence and a barcode sequence.

[0010] In some embodiments, the non-complementary region of the methylation specific primer comprises a restriction enzyme sequence and a biotinylated nucleotide sequence.

[0011] In some embodiments, the non-complementary region of the methylation specific primer comprises a rolling amplification sequence, a restriction enzyme sequence, and a biotinylated nucleotide sequence.

[0012] In some embodiments, one of the flanking arms is located on a 3' end of the methylation specific primer.

[0013] In some embodiments, one of the flanking arms is located on a 5' end of the methylation specific primer.

[0014] In some embodiments, generating the plurality of single-stranded circular nucleic acids comprises ligating the flanking arms of the methylation specific primers.

[0015] In some embodiments, the ligation of the flanking arms of the methylation specific primers is performed whilst the flanking arms are hybridised to the converted nucleic acid molecule.Attorney Docket No. HRG-030WO

[0016] In some embodiments, the converted nucleic acid molecules are converted from DNA.

[0017] In some embodiments, the DNA is cell-free DNA.

[0018] In some embodiments, the converted nucleic acid molecules are converted from RNA.

[0019] In some embodiments, the converted nucleic acid molecules were obtained by treating nucleic acids using bisulfite conversion to convert unmethylated cytosines to uracil.

[0020] In some embodiments, the converted nucleic acid molecules were obtained by treating nucleic acids using enzymatic conversion to convert unmethylated cytosines to uracil.

[0021] In some embodiments, the enzymatic conversion is selected from TET2 oxidation of cytosines and APOBEC conversion of cytosines.

[0022] In some embodiments, the sample comprises a blood sample, a stool sample, a urine sample, a mucous sample, or a saliva sample.

[0023] In some embodiments, the target sequence comprises at least a CpG island, or a portion thereof.

[0024] In some embodiments, the CpG island comprises a range of genomic locations shown in Table 1 or 2.

[0025] In some embodiments, enriching for the plurality of single-stranded circular nucleic acids comprises performing rolling circle amplification using the plurality of single-stranded circular nucleic acids.

[0026] In some embodiments, enriching for the plurality of single-stranded circular nucleic acids comprises capturing the plurality of single-stranded nucleic acids on solid supports.

[0027] In some embodiments, the solid supports comprise streptavidin-coated beads.

[0028] In some embodiments, the plurality of single-stranded nucleic acids comprise a biotinylated nucleotide sequence that are captured by the streptavidin-coated beads.

[0029] In some embodiments, the method further comprises: subsequent to enriching for the plurality of single -stranded circular nucleic acids, digesting the enriched plurality of single-stranded circular nucleic acids.

[0030] In some embodiments, digesting the enriched plurality of single-stranded circular nucleic acids comprises providing restriction enzymes to the enriched plurality of singlestranded circular nucleic acids.Attorney Docket No. HRG-030WO

[0031] In some embodiments, the restriction enzymes digest the enriched plurality of single-stranded circular nucleic acids at restriction enzyme sequences present in the enriched plurality of single-stranded circular nucleic acids.

[0032] In some embodiments, detecting the target sequence from the plurality of singlestranded circular nucleic acids comprises sequencing the plurality of single-stranded circular nucleic acids.

[0033] In some embodiments, sequencing the plurality of single -stranded circular nucleic acids comprises sequencing one or more barcode sequences present in the enriched plurality of single -stranded circular nucleic acids.

[0034] In some embodiments, detecting the target sequence from the plurality of single -stranded circular nucleic acids comprises performing one or more of qPCR, ddPCR, or microarray analysis.

[0035] In some embodiments, detecting the target sequence from the plurality of single -stranded circular nucleic acids comprises performing a gel blot analysis.

[0036] In one aspect, provided herein are complexes comprising: a converted nucleic acid molecule comprising a target sequence derived from a sequence comprising one or more methylated CpG sites; a methylation specific primer hybridised to the converted nucleic acid, the methylation specific primer comprising a non-complementary region and flanking arms that flank the non-complementary region, wherein a first flanking arm is hybridised to the target sequence of the converted nucleic acid molecule and a second flanking arm is hybridised to a sequence adjacent to the target sequence of the converted nucleic acid molecule.

[0037] In some embodiments, the non-complementary region of the methylation specific primer comprises a rolling amplification sequence.

[0038] In some embodiments, the non-complementary region of the methylation specific primer comprises a restriction enzyme sequence.

[0039] In some embodiments, the non-complementary region of the methylation specific primer comprises a barcode sequence.

[0040] In some embodiments, the non-complementary region of the methylation specific primer comprises a biotinylated nucleotide sequence.

[0041] In some embodiments, the non-complementary region of the methylation specific primer comprises a rolling amplification sequence and a barcode sequence.

[0042] In some embodiments, the non-complementary region of the methylation specific primer comprises a restriction enzyme sequence and a biotinylated nucleotide sequence.Attorney Docket No. HRG-030WO

[0043] In some embodiments, the non-complementary region of the methylation specific primer comprises a rolling amplification sequence, a restriction enzyme sequence, and a biotinylated nucleotide sequence.

[0044] In some embodiments, one of the flanking arms is located on a 3' end of the methylation specific primer.

[0045] In some embodiments, one of the flanking arms is located on a 5' end of the methylation specific primer.BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The foregoing and other objects, features and advantages of the invention will become apparent from the following description of preferred embodiments, as illustrated in the accompanying drawings. Like referenced elements identify common features in the corresponding drawings. The drawings are not necessarily to scale, with emphasis instead being placed on illustrating the principles of the present invention, in which:

[0047] It is noted that wherever practicable similar or like reference numbers may be used in the figures and may indicate similar or like functionality. For example, a letter after a reference numeral, such as “complementary region 310A,” indicates that the text refers specifically to the element having that particular reference numeral. A reference numeral in the text without a following letter, such as “complementary region 310” refers to any or all of the elements in the figures bearing that reference numeral (e.g. “complementary region 310” in the text refers to reference numerals “complementary region 310A” and / or “complementary region 310B” in the figures).

[0048] Figure (FIG.) 1A shows an example flow diagram for detecting target sequences, in accordance with an embodiment.

[0049] FIG. IB shows an exemplary scheme for performing rolling circle amplification.

[0050] FIG. 2A depicts an example conversion of nucleic acids, in accordance with an embodiment.

[0051] FIG. 2B shows the results of nitrite conversion on select nucleotides, in accordance with a second embodiment. Figure adapted from Li et al. (2022) Genome Biology 23: 122.

[0052] FIGs. 3A-3D show example primer designs, in accordance with an embodiment.

[0053] FIG. 4A depicts diagrams involving primers hybridized to a nucleic acid molecule, in accordance with an embodiment.Attorney Docket No. HRG-030WO

[0054] FIG. 4B depicts diagrams involving circularization of nucleic acids to generate a single-stranded circular DNA, in accordance with an embodiment.

[0055] FIG. 4C depicts a flow process for detecting the target sequence using the singlestranded circular DNA, in accordance with a first embodiment.

[0056] FIG. 4D depicts a flow process for detecting the target sequence using the singlestranded circular DNA, in accordance with a second embodiment.

[0057] FIG. 4E depicts a flow process for detecting the target sequence using the singlestranded circular DNA, in accordance with a third embodiment.

[0058] FIGs. 5A and 5B show an exemplary process for detecting target sequences using a primer including 1) primer site CRA Taq and 2) barcode sequence. This exemplary process includes steps for performing rolling circle amplification and sequencing.

[0059] FIGs. 6A and 6B show an exemplary process for detecting target sequences using a primer including 1) restriction enzyme site and 2) biotinylated nucleotide. This exemplary process includes steps for streptavidin capture, restriction enzyme digestion, and detection using qPCR / ddPCR / microarray.

[0060] FIGs. 7A and 7B show an exemplary process for detecting target sequences using a primer including 1) primer site CRA Taq, 2) restriction enzyme site and 3) biotinylated nucleotide. This exemplary process includes steps for streptavidin capture, rolling circle amplification, restriction enzyme digestion, and detection using a gel blot.DETAILED DESCRIPTIONDefinitions

[0061] The articles “a” and “an” are used herein to refer to one or to more than one (z. e. , to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0062] As used herein, the term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. “About” can mean a range of ±20%, ±10%, ±5%, or ±1% of a given value. The term “about” or “approximately” can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where a particular value is described in the application and claims, unless otherwise stated the term “about” meaning within anAttorney Docket No. HRG-030WOacceptable error range for the particular value can be assumed. The term “about” can have the meaning as commonly understood by one of ordinary skill in the art. The term “about” can refer to ±10%. The term “about” can refer to ±5%.

[0063] It should be understood that the expression of “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0064] As used herein, the term “biological sample,” or “sample” refers to any sample taken from a subject, which can reflect a biological state associated with the subject, and that includes cell free DNA. A biological sample can take any of a variety of forms, such as a liquid biopsy (e.g., blood, urine, stool, saliva, or mucous), or a tissue biopsy, or other solid biopsy. Examples of biological samples include, but are not limited to, blood, whole blood, plasma, serum, urine, cerebrospinal fluid, fecal, saliva, sweat, tears, pleural fluid, pericardial fluid, or peritoneal fluid of the subject. A biological sample can include any tissue or material derived from a living or dead subject. A biological sample can be a cell-free sample. A biological sample can comprise a nucleic acid (e.g., DNA or RNA) or a fragment thereof. The term “nucleic acid” can refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or any hybrid or fragment thereof. The nucleic acid in the sample can be a cell-free nucleic acid. A sample can be a liquid sample or a solid sample (e.g., a cell or tissue sample). A biological sample can be a bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, fluid from a hydrocele (e.g., of the testis), vaginal flushing fluids, pleural fluid, ascitic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, discharge fluid from the nipple, aspiration fluid from different parts of the body (e.g., thyroid, breast), etc. A biological sample can be a stool sample. In various embodiments, the majority of DNA in a biological sample that has been enriched for cell-free DNA (e.g., a plasma sample obtained via a centrifugation protocol) can be cell-free (e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the DNA can be cell-free). A biological sample can be treated to physically disrupt tissue or cell structure (e.g. , centrifugation and / or cell lysis), thus releasing intracellular components into a solution which can further contain enzymes, buffers, salts, detergents, and the like which can be used to prepare the sample for analysis.

[0065] As used herein, the terms “nucleic acid” and “nucleic acid molecule” are used interchangeably. The terms refer to nucleic acids of any composition form, such as deoxyribonucleic acid (DNA, e.g., complementary DNA (cDNA), genomic DNA (gDNA)Attorney Docket No. HRG-030WOand the like), and / or DNA analogs (e.g., containing base analogs, sugar analogs and / or a nonnative backbone and the like), all of which can be in single- or double-stranded form. Unless otherwise limited, a nucleic acid can comprise known analogs of natural nucleotides, some of which can function in a similar manner as naturally occurring nucleotides. A nucleic acid can be in any form useful for conducting processes herein (e.g., linear, circular, supercoiled, single-stranded, double -stranded and the like). A nucleic acid in some embodiments can be from a single chromosome or fragment thereof (e.g., a nucleic acid sample may be from one chromosome of a sample obtained from a diploid organism). In certain embodiments nucleic acids comprise nucleosomes, fragments or parts of nucleosomes or nucleosome-like structures. Nucleic acids can comprise protein (e.g., histones, DNA binding proteins, and the like). Nucleic acids analyzed by processes described herein can be substantially isolated and are not substantially associated with protein or other molecules. Nucleic acids can also include derivatives, variants and analogs of DNA synthesized, replicated or amplified from single-stranded (“sense” or “antisense,” “plus” strand or “minus” strand, “forward” reading frame or “reverse” reading frame) and double-stranded polynucleotides.Deoxyribonucleotides can include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. A nucleic acid may be prepared using a nucleic acid obtained from a subject as a template.

[0066] As used herein, the terms “template nucleic acid” and “template nucleic acid molecule(s)” are used interchangeably. The terms refer to nucleic acid that has been obtained from a sample and processed to form an immortalized library. The template nucleic acid can be nucleic acid obtained directly from the sample, or nucleic acid that is derived from that obtained directly from the sample. Examples of nucleic acid derived from a sample include DNA that has been reverse-transcribed from RNA obtained directly from a sample, or DNA that has be amplified from DNA obtained directly from a sample, for example, by PCR.

[0067] As used herein, the term “cell-free nucleic acids” refers to nucleic acid molecules that can be found outside cells, in bodily fluids such as blood, whole blood, plasma, serum, urine, cerebrospinal fluid, fecal, saliva, sweat, sweat, tears, pleural fluid, pericardial fluid, or peritoneal fluid of a subject. Cell-free nucleic acids originate from one or more healthy cells and / or from one or more cancer cells, or from non-human sources such bacteria, fungi, viruses. Examples of the cell-free nucleic acids include but are not limited to cell-free DNA (“cfDNA”), including mitochondrial DNA or genomic DNA, and cell-free RNA. In certain embodiments herein, instruments for assessing the quality of the cell-free nucleic acids, such as the TapeStation System from Agilent Technologies (Santa Clara, CA) can be used.Attorney Docket No. HRG-030WOConcentrating low-abundance cfDNA can be accomplished, for example using a Qubit™ Fluorometer from Thermofisher Scientific (Waltham, MA).

[0068] As used herein, the term “methylation” refers to a modification of a nucleic acid where a hydrogen atom on the pyrimidine ring of a cytosine base is converted to a methyl group, forming 5 -methylcytosine. Methylation can occur at dinucleotides of cytosine and guanine referred to herein as “CpG sites”. Methylation of cytosine can occur in cytosines in other sequence contexts, for example, 5'-CHG-3' and 5'-CHH-3', where H is adenine, cytosine or thymine. Cytosine methylation can also be in the form of 5-hydroxymethylcytosine. Methylation of DNA can include methylation of non-cytosine nucleotides, such as N6-methyladenine. Anomalous cfDNA methylation can be identified as hypermethylation or hypomethylation, both of which may be indicative of cancer status. As is well known in the art, DNA methylation anomalies (compared to healthy controls) can cause different effects, which may contribute to cancer.

[0069] Certain portions of a genome comprise regions with a high frequency of CpG sites. A CpG site is portion of a genome that has cytosine and guanine separated by only one phosphate group and is often denoted as “51— C — phosphate — G — 3'”, or “CpG” for short. Regions with a high frequency of CpG sites are commonly referred to as “CG islands” or “CGIs”. It has been found that certain CGIs and certain features of certain CGIs in tumor cells tend to be different from the same CGIs or features of the CGIs in healthy cells. Herein, such CGIS and features of the genome are referred to herein as “cancer informative CGIs”, which is defined and described in more detail below. An “informative CpG” can be specified by reference to a specific CpG site, or to a collection of one or more CpG sites by reference to a CG island that contains the collection. These cancer informative CGIs tend to have methylation patterns in tumor cells that are different from the methylation patterns in healthy cells. DNA fragments from other CGIs may not express such differences.

[0070] As used herein, “DNA methylation” in mammalian genomes can refer to the addition of a methyl group to position 5 of the heterocyclic ring of cytosine (e.g., to produce 5 -methylcytosine) among CpG dinucleotides. Methylation of cytosine can occur in cytosines in other sequence contexts, for example, 5'-CHG-3' and 5'-CHH-3', where H is adenine, cytosine or thymine. Cytosine methylation can also be in the form of 5-hydroxymethylcytosine. Methylation of DNA can include methylation of non-cytosine nucleotides, such as N6-methyladenine.

[0071] The phrase “target sequence” refers to a sequence of a nucleic acid derived from a sequence comprising one or more CpG sites (e.g., methylated or non-methylated CpG sites).Attorney Docket No. HRG-030WOFor example, the target sequence may be a sequence of a converted nucleic acid (e.g., where unmethylated cytosines have been converted to uracil and / or where methylated cytosines remain cytosines). In various embodiments, a target sequence includes one, two, three, four, five, six, seven, eight, nine, or ten CpG sites. In various embodiments, a target sequence includes one or more CpG sites within a region disclosed in Table 1 or Table 2. In particular embodiments, a target sequence includes five CpG sites within a region disclosed in Table 1 or Table 2. In particular embodiments, a target sequence includes five sequential CpG sites within a region disclosed in Table 1 or Table 2.

[0072] The phrase “candidate sequence” refers to a sequence of a nucleic acid derived from a sequence comprising one or more non-methylated CpG sites. For example, the candidate sequence may be a sequence of a converted nucleic acid (e.g., where unmethylated cytosines have been converted to uracil and / or where methylated cytosines remain cytosines). In various embodiments, a candidate sequence includes one, two, three, four, five, six, seven, eight, nine, or ten CpG sites. In various embodiments, a candidate sequence includes one or more CpG sites within a region disclosed in Table 1 or Table 2. In particular embodiments, a candidate sequence includes five CpG sites within a region disclosed in Table 1 or Table 2. In particular embodiments, a candidate sequence includes five sequential CpG sites within a region disclosed in Table 1 or Table 2.

[0073] The phrase “sequential CpG sites” refers to CpG sites within a range of genomic locations in which all CpG sites within the range of genomic locations are part of the sequential CpG sites. Sequential CpG sites include a neighboring CpG site i.e., a previous contiguous or next contiguous CpG site.

[0074] The phrase “unmethylated nucleic acid molecules” is broadly used to encompass nucleic acid molecules comprising sequences that include one or more unmethylated CpG sites and / or nucleic acid sequences derived from nucleic acid sequences that include one or more unmethylated CpG sites. For example, “unmethylated nucleic acid molecules” can refer to converted nucleic acid sequences (e.g., bisulfite-converted nucleic acid sequences) that are derived from cell-free DNA that include sequences with one or more unmethylated CpG sites.

[0075] As used herein, the term “amplifying” means performing an amplification reaction. In one aspect, an amplification reaction is “template-driven” in that base pairing of reactants, either nucleotides or oligonucleotides, have complements in a template polynucleotide that are required for the creation of reaction products. In one aspect, template-driven reactions are primer extensions with a nucleic acid polymerase, or oligonucleotide ligations with a nucleicAttorney Docket No. HRG-030WOacid ligase. Such reactions include, but are not limited to, polymerase chain reactions (PCRs), bisulfite-specific qPCR (qBSP), methylation-specific qPCR (qMSP), linear polymerase reactions, nucleic acid sequence -based amplification (NASBAs), rolling circle amplifications, and the like, disclosed in the following references, each of which are incorporated herein by reference herein in their entirety: Mullis et al., U.S. Pat. Nos.4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al., U.S. Pat. No. 5,210,015 (real-time PCR with “taqman” probes); Wittwer et al., U.S. Pat. No. 6,174,670; Kacian et al., U.S. Pat. No. 5,399,491 (“NASBA”); Uizardi, U.S. Pat. No. 5,854,033; Aono et al., Japanese patent publ. JP 4-262799 (rolling circle amplification); and the like. As used herein, bisulfite-specific PCR refers to PCR that amplifies converted DNA with no or limited methylation bias. As used herein, methylation-specific PCR refers to PCR that is methylation-specific and bi-sulfite specific, which amplifies converted methylated DNA. In one aspect, the amplification reaction is PCR. An amplification reaction may be a “real-time” amplification if a detection chemistry is available that permits a reaction product to be measured as the amplification reaction progresses, e.g., “real-time PCR”, or “real-time NASBA” as described in Ueone et al., Nucleic Acids Research, 26: 2150-2155 (1998), and like references.

[0076] The terms “fragment” or “segment”, as used interchangeably herein, refer to a portion of a larger polynucleotide molecule. A polynucleotide, for example, can be broken up, or fragmented into, a plurality of segments. Various methods of fragmenting nucleic acid are well known in the art. These methods may be, for example, either chemical or physical or enzymatic in nature. Enzymatic fragmentation may include partial degradation with a DNase; partial depurination with acid; the use of restriction enzymes; intron-encoded endonucleases; DNA-based cleavage methods, such as triplex and hybrid formation methods, that rely on the specific hybridization of a nucleic acid segment to localize a cleavage agent to a specific location in the nucleic acid molecule; or other enzymes or compounds which cleave a polynucleotide at known or unknown locations. Physical fragmentation methods may involve subjecting a polynucleotide to a high shear rate. High shear rates may be produced, for example, by moving DNA through a chamber or channel with pits or spikes, or forcing a DNA sample through a restricted size flow passage, e.g., an aperture having a cross sectional dimension in the micron or submicron range. Other physical methods include sonication and nebulization. Combinations of physical and chemical fragmentation methods may likewise be employed, such as fragmentation by heat and ion-mediated hydrolysis. See, e.g., Sambrook et al., “Molecular Cloning: A Eaboratory Manual,” 3rd Ed. Cold SpringAttorney Docket No. HRG-030WOHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) (“Sambrook et al.) which is incorporated herein by reference for all purposes. These methods can be optimized to digest a nucleic acid into fragments of a selected size range.

[0077] The terms “polymerase chain reaction” or “PCR”, as used interchangeably herein, mean a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Usually, the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument. Particular temperatures, durations at each step, and rates of change between steps depend on many factors that are well-known to those of ordinary skill in the art, e.g., exemplified by the following references: McPherson et al., editors, PCR: A Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively). For example, in a conventional PCR using Taq DNA polymerase, a double stranded target nucleic acid may be denatured at a temperature>90° C, primers annealed at a temperature in the range 50-75°C, and primers extended at a temperature in the range 72-78°C. The term “PCR” encompasses derivative forms of the reaction, including, but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, and the like. The particular format of PCR being employed is discernible by one skilled in the art from the context of an application. Reaction volumes can range from a few hundred nanoliters, e.g., 200 nL, to a few hundred pL, e.g., 200 pL. “Reverse transcription PCR,” or “RT-PCR,” means a PCR that is preceded by a reverse transcription reaction that converts a target RNA to a complementary single stranded DNA, which is then amplified, an example of which is described in Tecott et al., U.S. Pat. No. 5,168,038, the disclosure of which is incorporated herein by reference in its entirety. “Real-time PCR” means a PCR for which the amount of reaction product, i.e., amplicon, is monitored as the reaction proceeds. There are many forms of real-time PCR that differ mainly in the detection chemistries used for monitoring the reaction product, e.g., Gelfand et al., U.S. Pat. No. 5,210,015 (“taqman”); Wittwer et al., U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al., U.S. Pat. No. 5,925,517 (molecular beacons); the disclosures of which are hereby incorporated by reference herein in their entireties. Detection chemistries for real-time PCR are reviewed in Mackay et al., Nucleic Acids Research, 30: 1292-1305 (2002), which is also incorporatedAttorney Docket No. HRG-030WOherein by reference. “Nested PCR” means a two-stage PCR wherein the amplicon of a first PCR becomes the sample for a second PCR using a new set of primers, at least one of which binds to an interior location of the first amplicon. As used herein, “initial primers” in reference to a nested amplification reaction mean the primers used to generate a first amplicon, and “secondary primers” mean the one or more primers used to generate a second, or nested, amplicon. “Asymmetric PCR” means a PCR wherein one of the two primers employed is in great excess concentration so that the reaction is primarily a linear amplification in which one of the two strands of a target nucleic acid is preferentially copied. The excess concentration of asymmetric PCR primers may be expressed as a concentration ratio. Typical ratios are in the range of from 10 to 100. “Multiplexed PCR” means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture, e.g., Bernard et al., Anal. Biochem., 273: 221-228 (1999) (two-color real-time PCR). Usually, distinct sets of primers are employed for each sequence being amplified. Typically, the number of target sequences in a multiplex PCR is in the range of from 2 to 50, or from 2 to 40, or from 2 to 30. In particular embodiments, the number of target sequences in a multiplex PCR is about 4. “Quantitative PCR” means a PCR designed to measure the abundance of one or more specific target sequences in a sample or specimen. Quantitative PCR includes both absolute quantitation and relative quantitation of such target sequences. Quantitative measurements are made using one or more reference sequences or internal standards that may be assayed separately or together with a target sequence. The reference sequence may be endogenous or exogenous to a sample or specimen, and in the latter case, may comprise one or more competitor templates. Typical endogenous reference sequences include segments of transcripts of the following genes: -actin, GAPDH, 2 -microglobulin, ribosomal RNA, and the like. Techniques for quantitative PCR are well-known to those of ordinary skill in the art, as exemplified in the following references, which are incorporated by reference herein in their entireties: Freeman et al., Biotechniques, 26: 112-126 (1999); Becker-Andre et al., Nucleic Acids Research, 17: 9437-9447 (1989); Zimmerman et al., Biotechniques, 21: 268-279 (1996); Diviacco et al., Gene, 122: 3013-3020 (1992); and Becker-Andre et al., Nucleic Acids Research, 17: 9437-9446 (1989).

[0078] The term “primer” as used herein means an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. Extension of a primer is usually carried outAttorney Docket No. HRG-030WOwith a nucleic acid polymerase, such as a DNA or RNA polymerase. The sequence of nucleotides added in the extension process is determined by the sequence of the template polynucleotide. Usually, primers are extended by a DNA polymerase. Primers usually have a length in the range of from 14 to 40 nucleotides, or in the range of from 18 to 36 nucleotides. Primers are employed in a variety of nucleic amplification reactions, for example, linear amplification reactions using a single primer, or polymerase chain reactions, employing two or more primers. Guidance for selecting the lengths and sequences of primers for particular applications is well known to those of ordinary skill in the art, as evidenced by the following reference that is incorporated by reference herein in its entirety: Dieffenbach, editor, PCR Primer: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Press, New York, 2003).

[0079] As used herein, the term “subject” refers to any living or non-living organism, including but not limited to a human (e.g., a male human, female human, fetus, pregnant female, child, or the like), a non-human animal, a plant, a bacterium, a fungus or a protist. Any human or non-human animal can serve as a subject, including but not limited to mammal, reptile, avian, amphibian, fish, ungulate, ruminant, bovine (e.g., cattle), equine (e.g., horse), caprine and ovine (e.g., sheep, goat), swine (e.g., pig), camelid (e.g., camel, llama, alpaca), monkey, ape (e.g., gorilla, chimpanzee), ursid (e.g., bear), poultry, dog, cat, mouse, rat, fish, dolphin, whale and shark. In some embodiments, a subject is a male or female of any age (e.g., a man, a women or a child).

[0080] As used herein “selective enrichment of nucleic acid molecules” refers to the increased enrichment of nucleic acid molecules in relation to other nucleic acids. In various embodiments, selective enrichment refers to at least a fold enrichment of nucleic acid molecules relative to other nucleic acid molecules. Thus, in scenarios involving amplification and / or hybrid capture, selective enrichment of nucleic acid molecules does not require complete abatement or elimination of amplification and / or hybrid capture of other nucleic acid molecules. Rather, at least a fold increase in the amplification and / or hybrid capture of nucleic acid molecules is achieved in comparison to other nucleic acid molecules. In various embodiments, selective enrichment of nucleic acid molecules refers to at least a 2-fold increase, at least a 3-fold increase, at least a 4-fold increase, at least a 5-fold increase, at least a 6-fold increase, at least a 7-fold increase, at least a 8-fold increase, at least a 9-fold increase, at least a 10-fold increase, at least a 15-fold increase, at least a 20-fold increase, at least a 25 fold increase, at least a 50-fold increase, at least a 100-fold increase, at least a 200-Attorney Docket No. HRG-030WOfold increase, at least a 500-fold increase, or at least a 1000-fold increase of the nucleic acid molecules relative to other nucleic acids.

[0081] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0082] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press;Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0083] Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, and chemical analyses.

[0084] Throughout this specification and embodiments, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.Attorney Docket No. HRG-030WO

[0085] It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0086] Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting.

[0087] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0088] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to be inclusive of the numbers defining the range and to encompass any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10.

[0089] Where aspects or embodiments of the disclosure are described in terms of a Markush group or other grouping of alternatives, the present disclosure encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members. The present disclosure also envisages the explicit exclusion of one or more of any of the group members in an embodiment of the disclosure.

[0090] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. The materials, methods, and examples are illustrative only and not intended to be limiting.Overview

[0091] Disclosed herein are methods for performing differential enrichment of nucleic acid molecules e.g., enriching for nucleic acid molecules comprising a target sequence. Such methods are useful for enriching for a signal in a sample, such as a signal informative for determining presence or absence of cancer in the sample. For example, this enrichment technology can be used for methylation detection from bisulfite-converted DNA in qPCR, dPCR, hybridization capture, etc. Figure (FIG) 1A shows an example flow diagram forAttorney Docket No. HRG-030WOdetecting target sequences, in accordance with an embodiment. As shown in FIG. 1A, step 115 involves obtaining nucleic acid molecules from a sample (e.g., a sample from a subject). In various embodiments, a sample is any of a tissue sample, a blood sample, a stool sample, a urine sample, a mucous sample, or a saliva sample. In particular embodiments, a sample is a blood sample or a serum sample. In particular embodiments, a sample is a tissue sample. The sample can be obtained by the subject or by a third party, e.g., a medical professional.Examples of medical professionals include physicians, emergency medical technicians, nurses, first responders, psychologists, phlebotomists, medical physics personnel, nurse practitioners, surgeons, dentists, and any other medical professional as would be known to one skilled in the art. In various embodiments, the one or more samples can be obtained from the subject by a reference lab.

[0092] In various embodiments, the sample obtained from the subject is a liquid biopsy sample. In various embodiments, the liquid biopsy sample includes nucleic acid molecules. Example nucleic acid molecules include DNA or RNA. In particular embodiments, the nucleic acid molecules include cell-free DNA (cfDNA). In various embodiments, the cfDNA includes genomic sequences corresponding to CpG islands (CGIs) for which methylation states are informative for presence or absence of cancer. In various embodiments, the cfDNA can be derived from tumor cells and is referred to herein as circulating tumor DNA (ctDNA). In various embodiments, the nucleic acid molecules include a mixture of nucleic acid molecules that contain either methylated CpG sites or non-methylated CpG sites. For example, for a particular genomic region containing one or more CpG sites, the mixture of nucleic acid molecules includes a subset of nucleic acid molecules in which the one or more CpG sites are unmethylated and in a different subset of nucleic acid molecules in which the one or more CpG sites are partially or fully methylated.

[0093] Step 120 involves converting the nucleic acid molecules from the sample obtained from the subject. In various embodiments, converting the nucleic acid involves converting unmethylated nucleotides (e.g., cytosines) to another nucleotide (a “converted nucleotide,” as used herein) (e.g. uracil). In various embodiments, the converted nucleic acid molecules were obtained by treating nucleic acids using bisulfite conversion. In various embodiments, the converted nucleic acid molecules were obtained by treating the nucleic acids using enzymatic conversion. In various embodiments, the enzymatic conversion is selected from TET2 oxidation of cytosines and APOBEC conversion of cytosines. In various embodiments, methylated cytosines are protected from conversion (e.g., deamination) during the conversionAttorney Docket No. HRG-030WOstep. Further details of performing conversion of nucleic acid molecules (e.g., step 120) are described herein.

[0094] Although not shown in FIG. 1A, in various embodiments, after conversion of nucleic acids, the converted nucleic acids undergo library construction. In various embodiments, converted nucleic acids can undergo end-repairing, tailing of 3 ’ ends, and / or addition of library or sequencing adapters. In various embodiments, converted nucleic acids can undergo biotinylation (e.g., addition of biotin moieties to converted nucleic acids). In various embodiments, barcodes can be incorporated into converted nucleic acids, thereby enabling subsequent sample demultiplexing (e.g., demultiplexing to identify sources of converted nucleic acids or demultiplexing to identify a common source from converted nucleic acids). In various embodiments, one or more washes and / or selections can be performed to remove unwanted DNA fragments, such as single stranded DNA fragments, excess adapters, and other molecules. In particular embodiments, a solid-phase reversible immobilization (SPRI) selection is performed. As used herein, a “nucleic acid template” refers to a nucleic acid derived from the converted nucleic acid (e.g., any of a nucleic acid derived from a converted nucleic acid that underwent library construction, end-repairing, addition of library or sequencing adapters, barcode addition, or any combination thereof).

[0095] Step 130 involves providing one or more methylation specific primers to the converted nucleic acid molecules to generate one or more complexes comprising a methylation specific primer and a converted nucleic acid molecule, the methylation specific primer comprising flanking arms, at least one flanking arm hybridized to a target sequence of the converted nucleic acid molecule.

[0096] Step 140 involves ligating the flanking arms of methylation specific primers to generate a plurality of single-stranded circular nucleic acids.

[0097] Step 150 involves enrichment for the plurality of single-stranded circular nucleic acids. In various embodiments, enriching for the plurality of single-stranded circular nucleic acids comprises performing rolling circle amplification using the plurality of single-stranded circular nucleic acids on solid supports. In various embodiments, the solid supports comprise streptavidin-coated beads. In various embodiments, the plurality of single-stranded circular nucleic acids are captured by the streptavidin-coated beads.

[0098] Step 160 involves detecting the target sequence from the plurality of single-stranded circular nucleic acids. For example, step 160 may involve sequencing the target sequence.Attorney Docket No. HRG-030WOMethods for Enriching Target Nucleic Acid Sequencesa. Example Designs of Sequences

[0099] Disclosed herein are methylation specific primers that are useful for identifying target sequences (e.g., target methylation sequences). In various embodiments, methylation specific primers include at least a region designed to be complementary to a target sequence of a nucleic acid (e.g., a converted nucleic acid derived from cell free DNA with a sequence comprising one or more methylated CpG sites). The region designed to be complementary to a target sequence of a nucleic acid can, in various embodiments, be located on an arm of the methylation specific primer. In various embodiments, the region designed to be complementary to a target sequence of a nucleic acid is located on a 3 ’ end of the methylation specific primer. In various embodiments, the region designed to be complementary to a target sequence of a nucleic acid is located on a 5 ’ end of the methylation specific primer.

[0100] In various embodiments, methylation specific primers include a non-complementary region, such as a region that is non-complementary to the nucleic acid containing the target sequence. In various embodiments, the region designed to be complementary to a target sequence of a nucleic acid is positioned such that it flanks the non-complementary region (also referred to herein as a “flanking arm”). In various embodiments, methylation specific primers include two flanking arms that flank the non-complementary region, where each flanking arm is complementary to a corresponding sequence of the nucleic acid. For example, a first flanking arm can be complementary to the target sequence of the nucleic acid and a second flanking arm can be complementary to a different sequence of the nucleic acid. In various embodiments, the second flanking arm is complementary to a different sequence of the nucleic acid that is located adjacent or nearly adjacent to the target sequence of the nucleic acid. As referred to herein, “adjacent” refers to two sequences that are directly next to each other (e.g., no other nucleotides are located between the two sequences). As referred to herein, “nearly adjacent” refers to two sequences that have less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or 1 nucleotide between them.

[0101] In various embodiments, the region designed to be complementary to a target sequence of a nucleic acid is at least 90%, at least 95%, or 100% complementary to a target sequence, or a portion thereof. In various embodiments, the region designed to be complementary to a target sequence is at least 90%, at least 95%, or 100% complementary to a target sequence that includes one or more CpG sites within a region disclosed in Table 1 orAttorney Docket No. HRG-030WOTable 2. In various embodiments, the region designed to be complementary to a target sequence is at least 90%, at least 95%, or 100% complementary to a target sequence that includes two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more CpG sites within a region disclosed in Table 1 or Table 2. In various embodiments, the region designed to be complementary to a target sequence is at least 90%, at least 95%, or 100% complementary to a target sequence or a candidate sequences that includes two, three, four, five, six, seven, eight, nine, or ten CpG sites within a region disclosed in Table 1 or Table 2. In various embodiments, the region designed to be complementary to a target sequence is at least 90%, at least 95%, or 100% complementary to a target sequence that includes five sequential CpG sites within a region disclosed in Table 1 or Table 2. In various embodiments, the region designed to be complementary to a target sequence is 100% complementary to a target sequence that includes five sequential CpG sites within a region disclosed in Table 1 or Table 2.

[0102] In various embodiments, the region designed to be complementary to a target sequence is complementary to a target sequence that contains X CpG sites, where the X CpG sites are fully unmethylated. In various embodiments, the region designed to be complementary to a target sequence is complementary to a target sequence that contains X CpG sites, where at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the X CpG sites are unmethylated. As referred to herein, the term “K*n#” refers to a sequence having “*” CpG sites, whereof the CpG sites that are methylated. Therefore, the term “K5n5” refers to a sequence including 5 CpG sites in which 5 of the CpG sites are methylated. As another example, the term “K6n5” refers to a sequence including 6 CpG sites in which 5 of the CpG sites are methylated. In various embodiments, “*” can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In various embodiments,can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0103] In various embodiments, the region designed to be complementary to a target sequence can be fully complementary to a target sequence derived from a “K*n#” sequence. For example, the region designed to be complementary to a target sequence can be complementary to a target sequence derived from a K5n0 sequence (e.g., fully unmethylated sequence of 5 CpG sites). Thus, as each of the CpG sites of the K5n0 sequence is unmethylated, the resulting sequence corresponding to each CpG site may be “TG”, or a complement thereof. Thus, the region designed to be complementary to a target sequence can have a sequence that is complementary to each of the five “TG”, or complement thereof.Attorney Docket No. HRG-030WO

[0104] In various embodiments, the region designed to be complementary to a target sequence can be complementary to a target sequence derived from any of a KlnO sequence, a K2n0 sequence, a K3n0 sequence, a K4n0 sequence, a K5n0 sequence, a K6n0 sequence, a K7n0 sequence, a K8n0 sequence, a K9n0 sequence, or a KlOnO sequence. In various embodiments, the region designed to be complementary to a target sequence can be complementary to a target sequence derived from any of a K2nl sequence, a K3nl sequence, a K4nl sequence, a K5nl sequence, a K6nl sequence, a K7nl sequence, a K8nl sequence, a K9nl sequence, a KlOnl sequence, a K3n2 sequence, a K4n2 sequence, a K5n2 sequence, a K6n2 sequence, a K7n2 sequence, a K8n2 sequence, a K9n2 sequence, a K10n2 sequence, a K4n3 sequence, a K5n3 sequence, a K6n3 sequence, a K7n3 sequence, a K8n3 sequence, a K9n3 sequence, a K10n3 sequence, a K5n4 sequence, a K6n4 sequence, a K7n4 sequence, a K8n4 sequence, a K9n4 sequence, a K10n4 sequence, a K6n5 sequence, a K7n5 sequence, a K8n5 sequence, a K9n5 sequence, a K10n5 sequence, a K7n6 sequence, a K8n6 sequence, a K9n6 sequence, a K10n6 sequence, a K8n7 sequence, a K9n7 sequence, a K10n7 sequence, a K9n8 sequence, a K10n8 sequence, or a K10n9 sequence.

[0105] FIGs. 3A-3D show example primer designs, in accordance with an embodiment. In various embodiments, the non-complementary region of the methylation specific primer comprises a rolling amplification sequence. In various embodiments, the non-complementary region of the methylation specific primer comprises a restriction enzyme sequence. In various embodiments, the non-complementary region of the methylation specific primer comprises a barcode sequence. In various embodiments, the non-complementary region of the methylation specific primer comprises a biotinylated nucleotide sequence. In various embodiments, the non-complementary region of the methylation specific primer comprises a rolling amplification sequence and a barcode sequence. In various embodiments, the non-complementary region of the methylation specific primer comprises a restriction enzyme sequence and a biotinylated nucleotide sequence. In various embodiments, the non-complementary region of the methylation specific primer comprises a rolling amplification sequence, a restriction enzyme sequence, and a biotinylated nucleotide sequence.b. Exemplary Methods

[0106] FIG. 4A depicts diagrams involving primers hybridized to a nucleic acid molecule, in accordance with an embodiment. Step A begins with the converted nucleic acid molecule 415A. Step A shows the converted nucleic acid 415A after having performed step 120 shownAttorney Docket No. HRG-030WOin FIG. 1A. Here, the nucleic acid molecule 415A may include a target sequence 405 that is derived from a sequence comprising one or more methylated CpG sites. FIG. 4A shows two genomic sites (labeled as “m”) that are derived from corresponding CpG sites 402 that were previously methylated. Following conversion, the target sequence 405 can include a “CG,” or complement thereof, at each site corresponding to a CpG site. Here, one or more of the methylation specific primers 300 hybridizes to the target sequence 405 of the converted nucleic acid 415A. Step B begins when one or more complexes comprising a methylation specific primer 300 and a converted nucleic acid molecule 415A is formed as shown in step 130 of FIG. 1A. In Step B, the methylation specific primer comprises anon-complementary region 360 and flanking arms 310A and 310B that flank the non-complementary region, with a first flanking arm hybridized to the target sequence 405 of the converted nucleic acid molecule 415 A and a second flanking arm that is hybridized to a sequence adjacent to the target sequence of the converted nucleic acid molecule 415A.

[0107] FIG. 4B depicts diagrams involving circularization of nucleic acids to generate a single-stranded circular DNA, in accordance with an embodiment. Step C shows the ligation 408 of the flanking arms 310A and 310B of the methylation specific primers, as described in step 140 of FIG. 1A. In some embodiments, the ligation of the flanking arms of the methylation specific primers is performed while the flanking arms are hybridized to the converted nucleic acid molecule. Step D depicts the resultant single-stranded circular DNA 420, which contains the non-complementary region 360, as well as the flanking arms 310A and 310B.

[0108] FIG. 4C depicts a flow process for detecting the target sequence using the singlestranded circular DNA, in accordance with a first embodiment. Step 430 involves obtaining single-stranded circular DNA. Step 432 involves enriching the single-stranded circular DNA by performing rolling circle amplification. Step 434 involves sequencing the amplicons. Step 436 involves detecting the target sequence.

[0109] FIG. 4D depicts a flow process for detecting the target sequence using the singlestranded circular DNA, in accordance with a second embodiment. Step 440 involves obtaining single-stranded circular DNA. Step 442 involves enriching single-stranded circular DNA by capturing on solid supports. Step 444 involves decircularizing the enriched DNA. Step 446 involves detecting a target sequence using the decircularized DNA.

[0110] FIG. 4E depicts a flow process for detecting the target sequence using the singlestranded circular DNA, in accordance with a third embodiment. Step 450 involves obtaining single-stranded circular DNA. Step 452 involves enriching the single-stranded circular DNAAttorney Docket No. HRG-030WOby capturing on solid supports. Step 454 involves performing rolling circle amplification of the enriched single-stranded circular DNA. Step 456 involves digesting and generating fragments of amplicons. Step 458 involves detecting the target sequence using fragments of amplicons.c. Enrichment Steps

[0111] In certain embodiments, the target sequence or a subset of target sequences in the nucleic acid can be enriched using one or more additional enrichment steps. The one or more additional enrichments steps can be performed using any enrichment method known in the art. Non-limiting examples include hybrid capture, use of DNA-binding proteins to enrich a target sequence or a subset of target sequences, and nucleic acid amplification (e.g., polymerase chain reaction). In various embodiments, the additional enrichment step involves performing indexing PCR amplification e.g., using library adapters (e.g., P5 / P7 adapters). Thus, selective amplification nucleic acids with hybridized primer sequences can be performed using the library adapters. In contrast, nucleic acids in which primer sequences are incapable of hybridizing with do not undergo PCR amplification.

[0112] One or more additional enrichment steps can be performed before or after individual steps shown in FIG. 1A. For example, in certain embodiments, the method comprises a first step of depleting a first subset of nucleic acids (e.g., unmethylated nucleic acids or converted nucleic acids derived from unmethylated nucleic acids), thereby leaving a second subset of nucleic acids (e.g., methylated nucleic acids or converted nucleic acids derived from methylated nucleic acids). The method comprises a second step of subjecting nucleic acid sequences comprising the target sequence to one or more additional enrichment steps to enrich for at least a subset of the target sequences.

[0113] In certain embodiments, the method comprises a first step of subjecting a plurality of nucleic acid molecules that include target sequences to an enrichment step to enrich for the target sequences. The method further comprises a second step of subjecting the plurality of nucleic acid molecules to the depletion method disclosed herein, which depletes a first subset of nucleic acids (e.g., unmethylated nucleic acids or converted nucleic acids derived from unmethylated nucleic acids) thereby leaving a second subset of nucleic acids (e.g., methylated nucleic acids or converted nucleic acids derived from methylated nucleic acids).

[0114] In certain embodiments, a target sequence or a subset of target sequences in the nucleic acid can be enriched by subjecting the nucleic acid comprising the target sequence or the subset of target sequences to hybrid capture. In hybrid capture, labeled (e.g.,Attorney Docket No. HRG-030WObiotinylated) capture probes that can bind to one or more target sequences or subsets of target sequences are exposed to the nucleic acid comprising the one or more target sequences. The capture probes are specific to a sequence of interest, for example, a methylation pattern of interest that can be detected as a bisulfite-converted epitype. Examples of such hybrid capture probe sets include the KAPA HyperPrep KAPA HyperCap Workflow with HyperChoice Probes, Twist Bioscience Twist Fast Hybridization Custom Target Enrichment Panel, Integrated DNA technologies xGen Custom Hybridization Capture Panel, and SeqCAP Epi Enrichment System from Roche Diagnostics (Pleasanton, CA).Example Nucleic Acids and Methods for Converting Nucleic Acids

[0115] As discussed herein, step 120 in FIG. 1A involves converting nucleic acid molecules from an obtained sample. In various embodiments, converting nucleic acid molecules includes treating the nucleic acid molecules to capture methylation modifications. In various embodiments, converting nucleic acid molecules involves converting one or more unmethylated nucleotides (e.g., cytosines) to another nucleotide (a “converted nucleotide”, as used herein), e.g., using chemical or enzymatic means. In certain embodiments, one or more unmethylated cytosines are converted to a nucleotide that pairs with adenine (e.g., the unmethylated cytosine may be converted to uracil). In certain embodiments, one or more unmethylated adenines are converted to a base that pairs with cytosine (e.g., the unmethylated adenine may be converted to inosine (I)). In certain embodiments, one or more methylated cytosines (e.g., a 5 -methylcytosine (5mC)) is converted to a thymine, which pairs with adenine. In certain embodiments, methylated cytosines are protected from conversion (e.g., deamination) during the conversion step.

[0116] After a nucleic acid has been treated to convert unmethylated, or, in some cases, methylated nucleotides, into another nucleotide, the nucleic acid may be amplified. During amplification, the converted nucleotide pairs with its complementary nucleotide, and in the next round of amplification, the complementary nucleotide pairs with a replacement nucleotide. For example, following the conversion of an unmethylated cytosine to a uracil, the nucleic acid may be amplified such that an adenine pairs with the uracil in the first round of replication, and in the second round of replication, the adenine pairs with a thymine. Accordingly, the thymine replaces the uracil in the original nucleic acid sequence, and is referred to herein as a “replacement nucleotide”.

[0117] In certain aspects, conversion of the nucleic acids involves selectively deaminating nucleotides. FIG. 2A depicts an example conversion of nucleic acids, in accordance with an embodiment. Selective deamination refers to a process in which unmethylated cytosineAttorney Docket No. HRG-030WOresidues are selectively deaminated over methylated cytosine (5 -methylcytosine) residues. In certain embodiments, deamination of cytosine forms uracil, effectively inducing a C to T point mutation to allow for detection of methylated cytosines. Methods of deaminating cytosine are known in the art, and include chemical conversion (e.g., bisulfite conversion) and enzymatic conversion. In certain embodiments, the enzymatic conversion comprises subjecting the nucleic acid to TET2, which oxidizes methylated cytosines, thereby protecting them, and subsequent exposure to APOBEC, which converts unprotected (i.e., unmethylated) cytosines to uracils.

[0118] In some embodiments, the conversion, for example, bisulfite conversion or enzymatic conversion, uses commercially available kits. Bisulfite conversion can be performed using commercially available technologies, such as EZ DNA Methylation-Gold, EZ DNAMethylation-Direct or an EZ DNAMethylation-Lighting kit (Zymo Research Corp (Irvine, California)) or EpiTect Fast available from Qiagen (Germantown, MD). In another example a kit such as APOBECSeq (NEBiolabs) or One Step qMethyl-PCR Kit (Zymo Research Corp (Irvine, California)) is used.a. Source of Nucleic Acids

[0119] Nucleic acids used in the methods described herein can be derived from any source, such as a sample taken from the environment or from a subject (e.g., a human subject). A biological sample can be treated to physically disrupt tissue or cell structure (e.g., centrifugation and / or cell lysis), thus releasing intracellular components into a solution which can further contain enzymes, buffers, salts, detergents, and the like which can be used to prepare the sample for analysis. A biological sample can take any of a variety of forms, such as a liquid biopsy (e.g., blood, urine, stool, saliva, or mucous), or a tissue biopsy, or other solid biopsy. Examples of biological samples include, but are not limited to, blood, whole blood, plasma, serum, urine, cerebrospinal fluid, fecal, saliva, sweat, tears, pleural fluid, pericardial fluid, or peritoneal fluid of the subject. A biological sample can include any tissue or material derived from a living or dead subject. A biological sample can be a cell-free sample. A sample can be a liquid sample or a solid sample (e.g., a cell or tissue sample). A biological sample can be a bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, fluid from a hydrocele (e.g., of the testis), vaginal flushing fluids, pleural fluid, ascitic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, discharge fluid from the nipple, aspiration fluid from different parts of the body (e.g., thyroid, breast), etc.Attorney Docket No. HRG-030WO

[0120] The nucleic acid can be of any composition form, such as deoxyribonucleic acid (DNA, e.g., complementary DNA (cDNA), genomic DNA (gDNA) and the like), and / or DNA analogs (e.g., containing base analogs, sugar analogs and / or a non-native backbone and the like), and / or ribonucleic acid (RNA) and / or RNA analogs, all of which can be in single-or double -stranded form. In certain embodiments, single -stranded nucleic acids can be made double stranded prior to cutting with an enzyme. Unless otherwise limited, a nucleic acid can comprise known analogs of natural nucleotides, some of which can function in a similar manner as naturally occurring nucleotides. A nucleic acid can be in any form useful for conducting processes herein (e.g., linear, circular, supercoiled, single-stranded, doublestranded and the like). A nucleic acid in some embodiments can be from a single chromosome or fragment thereof (e.g., a nucleic acid sample may be from one chromosome of a sample obtained from a diploid organism). In certain embodiments nucleic acids comprise nucleosomes, fragments or parts of nucleosomes or nucleosome-like structures. Nucleic acids can comprise protein (e.g., histones, DNA binding proteins, and the like). Nucleic acids analyzed by processes described herein can be substantially isolated and are not substantially associated with protein or other molecules. Nucleic acids can also include derivatives, variants and analogs of DNA synthesized, replicated or amplified from singlestranded (“sense” or “antisense,” “plus” strand or “minus” strand, “forward” reading frame or “reverse” reading frame) and double -stranded polynucleotides. Deoxyribonucleotides can include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. A nucleic acid may be prepared using a nucleic acid obtained from a subject as a template.

[0121] In certain embodiments, the nucleic acid is a cell-free nucleic acid, which can be found in bodily fluids such as blood, whole blood, plasma, serum, urine, cerebrospinal fluid, fecal, saliva, sweat, sweat, tears, pleural fluid, pericardial fluid, or peritoneal fluid of a subject. In certain embodiments, a plasma sample can be used directly in the methods disclosed herein (for example, in the cutting step), without prior purification or isolation of nucleic acids in the plasma. Cell-free nucleic acids originate from one or more healthy cells and / or from one or more cancer cells, or from non-human sources such bacteria, fungi, viruses. Examples of the cell-free nucleic acids include but are not limited to cell-free DNA (“cfDNA”), including mitochondrial DNA or genomic DNA, and cell-free RNA. In certain embodiments herein, instruments for assessing the quality of the cell-free nucleic acids, such as the TapeStation System from Agilent Technologies (Santa Clara, CA) can be used.Concentrating low-abundance cfDNA can be accomplished, for example using a Qubit Fluorometer from Thermofisher Scientific (Waltham, MA).Attorney Docket No. HRG-030WO

[0122] In various embodiments, the majority of DNA in a biological sample that has been enriched for cell-free DNA (e.g., a plasma sample obtained via a centrifugation protocol) can be cell-free (e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the DNA can be cell-free).

[0123] A methylated nucleic acid is a nucleic acid having a modification in which a hydrogen atom on the pyrimidine ring of a cytosine base is converted to a methyl group, forming 5-methylcytosine. Methylation can occur at dinucleotides of cytosine and guanine referred to herein as “CpG sites”, which can be a target for enrichment. Methylation of cytosine can occur in cytosines in other sequence contexts, for example, 5'-CHG-3' and 5'-CHH-3', where H is adenine, cytosine or thymine. Cytosine methylation can also be in the form of 5 -hydroxymethylcytosine. Methylation of DNA can include methylation of noncytosine nucleotides, such as N6-methyladenine (6mA). Anomalous cfDNA methylation can be identified as hypermethylation or hypomethylation, both of which may be indicative of cancer status. As is well known in the art, DNA methylation anomalies (compared to healthy controls) can cause different effects, which may contribute to cancer.

[0124] In certain embodiments, the nucleic acid comprises a CpG site (i.e., cytosine and guanine separated by only one phosphate group). In certain embodiments, the nucleic acid comprises a CpG island (also referred to as a “CG islands” or “CGI”) or a portion thereof, which is the target for enrichment. Because certain CGIs and certain features of certain CGIs in tumor cells tend to be different from the same CGIs or features of the CGIs in healthy cells, detection of such CGIs can be informative of a health condition. In certain embodiments, the CGI is a “cancer informative CGIs”, which is defined and described in more detail below. In certain embodiments, the CpG is an “informative CpG”, e.g., a “cancer informative CGI”. Such CGIs may have methylation patterns in tumor cells that are different from the methylation patterns in healthy cells. Accordingly, detection of a cancer informative CGI can be informative regarding a subject’s risk of developing cancer or can be indicative that the subject has cancer. Exemplary cancer informative CGIs, which can be target sequences as described herein, are identified in, e.g., Table 1 of U.S. Patent Publication 2020 / 0109456A1 and Tables 2 and 3 ofWO2022 / 133315, each of which are hereby incorporated by reference in its entirety. Further exemplary cancer informative CGIs are shown in Tables 1 and 2 included herein.

[0125] In certain aspects, the nucleic acids of the invention have been treated to convert one or more unmethylated nucleotides (e.g., cytosines) to another nucleotide (a “converted nucleotide”, as used herein, such as a uracil), for example, prior to amplification. In certainAttorney Docket No. HRG-030WOembodiments, one or more unmethylated cytosines are converted to a nucleotide that pairs with adenine (e.g., the unmethylated cytosine may be converted to uracil). In certain embodiments, one or more unmethylated adenines are converted to a base that pairs with cytosine (e.g., the unmethylated adenine may be converted to inosine (I)). In certain embodiments, one or more methylated cytosines (e.g., a 5 -methylcytosine (5mC)) is converted to a thymine, which pairs with adenine. In certain embodiments, methylated cytosines are protected from conversion (e.g., deamination) during the conversion step.

[0126] After a nucleic acid has been treated to convert unmethylated, or, in some cases, methylated nucleotides, into another nucleotide, the nucleic acid may be amplified. During amplification, the converted nucleotide pairs with its complementary nucleotide, and in the next round of amplification, the complementary nucleotide pairs with a replacement nucleotide. For example, following the conversion of an unmethylated cytosine to a uracil, the nucleic acid may be amplified such that an adenine pairs with the uracil in the first round of replication, and in the second round of replication, the adenine pairs with a thymine. Accordingly, the thymine replaces the uracil in the original nucleic acid sequence, and is referred to herein as a “replacement nucleotide”.b. Bisulfite conversion

[0127] Bisulfite conversion is performed on DNA by denaturation using high heat, preferential deamination (at an acidic pH) of unmethylated cytosines, which are then converted to uracil by desulfonation (at an alkaline pH). Methylated cytosines remain unchanged on the single-stranded DNA (ssDNA) product.

[0128] In some embodiments the methods include treatment of the sample with bisulfite (e.g., sodium bisulfite, potassium bisulfite, ammonium bisulfite, magnesium bisulfite, sodium metabisulfite, potassium metabisulfite, ammonium metabisulfite, magnesium metabisulfite and the like). Unmethylated cytosine is converted to uracil through a three-step process during sodium bisulfite modification. As shown in FIG. 2A, the steps are sulphonation to convert cytosine to cytosine sulphonate, deamination to convert cytosine sulphonate to uracil sulphonate and alkali desulphonation to convert uracil sulphonate to uracil. Conversion on methylated cytosine is much slower and is not observed at significant levels in a 4-16 hour reaction. (See Clark et al., Nucleic Acids Res., 22(15):2990-7 (1994).) If the cytosine is methylated it will remain a methylated cytosine. If the cytosine is unmethylated it will be converted to uracil. When the modified strand is copied, for example, through extension of a locus specific primer, a random or degenerate primer or a primer to an adaptor, a G will beAttorney Docket No. HRG-030WOincorporated in the interrogation position (opposite the C being interrogated) if the C was methylated and an A will be incorporated in the interrogation position if the C was unmethylated and converted to U. When the double stranded extension product is amplified those Cs that were converted to Us and resulted in incorporation of A in the extended primer will be replaced by Ts during amplification. Those Cs that were not converted (i.e., the methylated Cs) and resulted in the incorporation of G will be replaced by unmethylated Cs during amplification.c. Enzymatic conversion

[0129] In certain embodiments, the enzymatic treatment with a cytidine deaminase enzyme is used to convert cytosine to uracil. Enzymatic conversion can include an oxidation step, in which Tet methylcytosine dioxygenase 2 (TET2) catalyzes the oxidation of 5mC to 5hmC to protect methylated cytosines from conversion by subsequent exposure to a cytidine deaminase. Other protection steps known in the art can be used in addition to or in place of oxidation by TET2. After the oxidation step, the nucleic acid is treated with the cytidine deaminase to convert one or more unmethylated cytosines to uracils. As with bisulfite conversion, when the modified strand is copied, a G will be incorporated in the interrogation position (opposite the C being interrogated) if the C was methylated and an A will be incorporated in the interrogation position if the C was unmethylated. When the double stranded extension product is amplified those Cs that were converted to Us and resulted in incorporation of A in the extended primer will be replaced by Ts during amplification. Those Cs that were not modified and resulted in the incorporation of G will remain as C.

[0130] In certain embodiments the cytidine deaminase may be APOBEC. In certain embodiments the cytidine deaminase includes activation induced cytidine deaminase (AID) and apolipoprotein B mRNA editing enzymes, catalytic polypeptide -like (APOBEC). In certain embodiments, the APOBEC enzyme is selected from the human APOBEC family consisting of: APOBEC-1 (Apol), APOBEC-2 (Apo2), AID, APOBEC-3A, -3B, -3C, -3DE, -3F, -3G, -3H and APOBEC-4 (Apo4). In certain embodiments, the APOBEC enzyme is APOBEC-seq.d. Nitrite Conversion

[0131] In certain embodiments, nitrite treatment is used to deaminate adenine and cytosine. As shown in FIG. 2B, deamination of an A results in conversion to an inosine (I), which is read by a polymerase as a G, whereas deamination of a methylated A (W-mcthyladcnincAttorney Docket No. HRG-030WO(6mA)) results in a nitrosylated 6mA (6mA-N0), which causes the base to be read by a polymerase as an A. Deamination of a C results in conversion to a uracil, which is read by a polymerase as a T, whereas deamination of a A4-methylcytosine (4mC) to 4mC-N0 or a 5-methylcytosine (5mC) to a T causes the base to be read by a polymerase as a C or a T, respectively. For 5mC bases, the C to T ratio at the 5mC position is about 40% higher than other cytosine positions, allowing 5mC to be differentiated from C. (See, Li et al. (2022) Genome Biology 23:122.)EXAMPLES

[0132] Practice of embodiments disclosed herein will be more fully understood from the foregoing examples, which are presented herein for illustrative purposes only, and should not be construed as limiting the invention in any way.Example 1 - General Methods for Detecting Target Sequences

[0133] Reference is made to FIGs. 5A and 5B, which show an exemplary process for detecting target sequences using a primer including 1) primer site CRA Taq and 2) barcode sequence. This exemplary process includes steps for performing rolling circle amplification and sequencing.

[0134] Nucleic acids with various methylation patterns are obtained. The nucleic acids undergo bisulfite conversion. Unmethylated cytosines are converted to uracil whereas methylated cytosines remain as cytosine nucleotides. Following conversion, nucleic acids undergo amplification, resulting in the presence of thymine nucleotides (T) in the place of the uracil nucleotides.

[0135] Methylation specific primers are provided to the nucleic acids. As shown in FIG.5 A, the methylation specific primers include a non-complementary region including 1) Primer site CRA Taq (e.g., a rolling amplification sequence) and 2) a barcode target (e.g., a barcode sequence). The methylation specific primer further includes flanking arms at the 3’ and 5’ ends. As shown in FIG. 5A, the 3’ end of the methylation specific primer includes a flanking arm labeled as “Methylation regions”. Here, the methylation regions has a sequence that is designed to be complementary to the sequence of the nucleic acid that underwent bisulfite conversion. Additionally, the 5’ end of the methylation specific primer includes a flanking arm with a sequence that is designed to be complementary to another sequence of the nucleic acid that underwent bisulfite conversion.

[0136] As shown in FIG. 5A, two different methylation specific primers are provided, thereby hybridizing with two different target sequences of the nucleic acid. The flanking armsAttorney Docket No. HRG-030WO(3 ’ and 5 ’ ends) of the methylation specific primers hybridize with corresponding sequences on the nucleic acid. The non-complementary region of the methylation specific primers circularize and are not bound to the nucleic acid. As shown in FIG. 5A, the flanking arms of the methylation specific primers hybridize with adjacent sequences on the nucleic acid.

[0137] The flanking arms of the methylation specific primers undergo ligation while hybridized to the sequences on the nucleic acid. The methylation specific primers are released, thereby generating single-stranded circular DNA.

[0138] Referring next to FIG. 5B, the single-stranded circular DNA undergoes rolling circle amplification. A Phi29 TaQ polymerase synthesizes a long sequence from the singlestranded circular DNA. The resulting long sequence includes tandem repeats and further includes the barcode sequence.

[0139] Different long sequences from different samples are pooled and undergo sequencing (e.g., nano-pore sequencing). Using the sequenced barcodes, the resulting sequence reads undergo assembly and analysis to identify target sequences (e.g., methylation patterns).

[0140] Reference is next made to FIGs. 6A and 6B, which show an exemplary process for detecting target sequences using a primer including 1) restriction enzyme site and 2) biotinylated nucleotide. This exemplary process includes steps for streptavidin capture, restriction enzyme digestion, and detection using qPCR / ddPCR / microarray.

[0141] Nucleic acids with various methylation patterns are obtained. The nucleic acids undergo bisulfite conversion. Unmethylated cytosines are converted to uracil whereas methylated cytosines remain as cytosine nucleotides. Following conversion, nucleic acids undergo amplification, resulting in the presence of thymine nucleotides (T) in the place of the uracil nucleotides.

[0142] Methylation specific primers are provided to the nucleic acids. As shown in FIG.6 A, the methylation specific primers include a non-complementary region including 1) restriction enzyme site (ATTC) (also referred to herein as a restriction enzyme sequence) and 2) a biotinylated nucleotide (e.g., a biotinylated nucleotide sequence). The methylation specific primer further includes flanking arms at the 3’ and 5’ ends. As shown in FIG. 6A, the 3 ’ end of the methylation specific primer includes a flanking arm labeled as “Methylation regions”. Here, the methylation regions has a sequence that is designed to be complementary to the sequence of the nucleic acid that underwent bisulfite conversion. Additionally, the 5’ end of the methylation specific primer includes a flanking arm with a sequence that is designed to be complementary to another sequence of the nucleic acid that underwent bisulfite conversion.Attorney Docket No. HRG-030WO

[0143] As shown in FIG. 6A, two different methylation specific primers are provided, thereby hybridizing with two different target sequences of the nucleic acid. The flanking arms (3’ and 5’ ends) of the methylation specific primers hybridize with corresponding sequences on the nucleic acid. The non-complementary region of the methylation specific primers circularize and are not bound to the nucleic acid. As shown in FIG. 6A, the flanking arms of the methylation specific primers hybridize with adjacent sequences on the nucleic acid.

[0144] The flanking arms of the methylation specific primers undergo ligation while hybridized to the sequences on the nucleic acid. The methylation specific primers are released, thereby generating single-stranded circular DNA.

[0145] Referring next to FIG. 6B, the single-stranded circular DNA undergoes enrichment by capturing the single -stranded circular DNA on streptavidin-coated beads. The beads are washed to remove non-bound nucleic acids. The enriched single-stranded circular DNA are released from the beads and undergo de-circularization through digestion. The enriched single-stranded circular DNA are exposed to restrict enzymes which cleave at restriction enzyme sequences that are present in the enriched single -stranded circular DNA.

[0146] Target sequences are detected in the digested nucleic acids by primer specific primers for the target sequences. By performing quantitative PCR, droplet digital PCR, or microarray analysis, the presence and / or quantity of the target sequences are determined.

[0147] FIGs. 7A and 7B show an exemplary process for detecting target sequences using a primer including 1) primer site CRA Taq, 2) restriction enzyme site and 3) biotinylated nucleotide. This exemplary process includes steps for streptavidin capture, rolling circle amplification, restriction enzyme digestion, and detection using a gel blot.Attorney Docket No. HRG-030WOTablesTable 1. Genomic ranges of low background regions including a plurality of CpG sites mapped to human genome, hgl9. The “Genomic Coordinate Start” and “Genomic Coordinate End” columns indicate the beginning and end, respectively of a range of genomic locations within a chromosome (“Chrom.”).Attorney Docket No. HRG-030WOTable 2: Exemplary Target RegionsHuman universal genomic locations and CpG sites mapped to human genome, hg38. The “Start” and “End” columns indicate the beginning and end, respectively of a range of a target region within a chromosome (“Chrom.”).Attorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WOAttorney Docket No. HRG-030WO

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for detecting a target sequence, the method comprising:obtaining converted nucleic acid molecules derived from a sample;providing one or more methylation specific primers to the converted nucleic acid molecules to generate one or more complexes comprising a methylation specific primer and a converted nucleic acid molecule, the methylation specific primer comprising a non-complementary region and flanking arms that flank the non- complementary region, at least one flanking arm hybridized to a target sequence of the converted nucleic acid molecule;generating a plurality of single-stranded circular nucleic acids using at least the methylation specific primers;enriching for the plurality of single-stranded circular nucleic acids; anddetecting the target sequence from the plurality of single -stranded circular nucleic acids.

2. The method of claim 1, wherein the non-complementary region of the methylation specific primer comprises a rolling amplification sequence.

3. The method of claim 1 or 2, wherein the non-complementary region of the methylation specific primer comprises a restriction enzyme sequence.

4. The method of claim 1 or 2, wherein the non-complementary region of the methylation specific primer comprises a barcode sequence.

5. The method of claim 1 or 2, wherein the non-complementary region of the methylation specific primer comprises a biotinylated nucleotide sequence.

6. The method of claim 1 or 2, wherein the non-complementary region of the methylation specific primer comprises a rolling amplification sequence and a barcode sequence.

7. The method of claim 1 or 2, wherein the non-complementary region of the methylation specific primer comprises a restriction enzyme sequence and a biotinylated nucleotide sequence.

8. The method of claim 1 or 2, wherein the non-complementary region of the methylation specific primer comprises a rolling amplification sequence, a restriction enzyme sequence, and a biotinylated nucleotide sequence.Attorney Docket No. HRG-030WO9. The method of any one of claims 1-8, wherein one of the flanking arms is located on a 3 ’ end of the methylation specific primer.

10. The method of any one of claims 1-9, wherein one of the flanking arms is located on a 5 ’ end of the methylation specific primer.

11. The method of any one of claims 1-10, wherein generating the plurality of singlestranded circular nucleic acids comprises ligating the flanking arms of the methylation specific primers.

12. The method of claim 11, wherein the ligation of the flanking arms of the methylation specific primers is performed while the flanking arms are hybridized to the converted nucleic acid molecule.

13. The method of any one of claims 1-12, wherein the converted nucleic acid molecules are converted from DNA.

14. The method of claim 13, wherein the DNA is cell-free DNA.

15. The method of any one of claims 1-12, wherein the converted nucleic acid molecules are converted from RNA.

16. The method of any one of claims 1-15, wherein the converted nucleic acid molecules were obtained by treating nucleic acids using bisulfite conversion to convert unmethylated cytosines to uracil.

17. The method of any one of claims 1-15, wherein the converted nucleic acid molecules were obtained by treating nucleic acids using enzymatic conversion to convert unmethylated cytosines to uracil.

18. The method of claim 17, wherein the enzymatic conversion is selected from TET2 oxidation of cytosines and APOBEC conversion of cytosines.

19. The method of any one of claims 1-18, wherein the sample comprises a blood sample, a stool sample, a urine sample, a mucous sample, or a saliva sample.

20. The method of any one of claims 1-19, wherein the target sequence comprises at least a CpG island, or a portion thereof.

21. The method of claim 20, wherein the CpG island comprises a range of genomic locations shown in Table 1 or 2.

22. The method of any one of claims 1-21, wherein enriching for the plurality of singlestranded circular nucleic acids comprises performing rolling circle amplification using the plurality of single-stranded circular nucleic acids.Attorney Docket No. HRG-030WO23. The method of any one of claims 1-21, wherein enriching for the plurality of singlestranded circular nucleic acids comprises capturing the plurality of single-stranded nucleic acids on solid supports.

24. The method of claim 23, wherein the solid supports comprise streptavidin-coated beads.

25. The method of claim 24, wherein the plurality of single-stranded nucleic acids comprise a biotinylated nucleotide sequence that are captured by the streptavidin-coated beads.

26. The method of any one of claims 1-25, further comprising:subsequent to enriching for the plurality of single-stranded circular nucleic acids, digesting the enriched plurality of single-stranded circular nucleic acids.

27. The method of claim 26, wherein digesting the enriched plurality of single -stranded circular nucleic acids comprises providing restriction enzymes to the enriched plurality of single-stranded circular nucleic acids.

28. The method of claim 27, wherein the restriction enzymes digest the enriched plurality of single -stranded circular nucleic acids at restriction enzyme sequences present in the enriched plurality of single -stranded circular nucleic acids.

29. The method of any one of claims 1-28, wherein detecting the target sequence from the plurality of single-stranded circular nucleic acids comprises sequencing the plurality of single-stranded circular nucleic acids.

30. The method of claim 29, wherein sequencing the plurality of single-stranded circular nucleic acids comprises sequencing one or more barcode sequences present in the enriched plurality of single-stranded circular nucleic acids.

31. The method of any one of claims 1-28, wherein detecting the target sequence from the plurality of single-stranded circular nucleic acids comprises performing one or more of qPCR, ddPCR, or microarray analysis.

32. The method of any one of claims 1-28, wherein detecting the target sequence from the plurality of single-stranded circular nucleic acids comprises performing a gel blot analysis.

33. A complex comprising :a converted nucleic acid molecule comprising a target sequence derived from a sequence comprising one or more methylated CpG sites;Attorney Docket No. HRG-030WOa methylation specific primer hybridized to the converted nucleic acid, the methylation specific primer comprising a non-complementary region and flanking arms that flank the non-complementary region,wherein a first flanking arm is hybridized to the target sequence of the converted nucleic acid molecule and a second flanking arm is hybridized to a sequence adjacent to the target sequence of the converted nucleic acid molecule.

34. The complex of claim 33, wherein the non-complementary region of the methylation specific primer comprises a rolling amplification sequence.

35. The complex of claim 33, wherein the non-complementary region of the methylation specific primer comprises a restriction enzyme sequence.

36. The complex of claim 33, wherein the non-complementary region of the methylation specific primer comprises a barcode sequence.

37. The complex of claim 33, wherein the non-complementary region of the methylation specific primer comprises a biotinylated nucleotide sequence.

38. The complex of claim 33, wherein the non-complementary region of the methylation specific primer comprises a rolling amplification sequence and a barcode sequence.

39. The complex of claim 33, wherein the non-complementary region of the methylation specific primer comprises a restriction enzyme sequence and a biotinylated nucleotide sequence.

40. The complex of claim 33, wherein the non-complementary region of the methylation specific primer comprises a rolling amplification sequence, a restriction enzyme sequence, and a biotinylated nucleotide sequence.

41. The complex of any one of claims 33-40, wherein one of the flanking arms is located on a 3 ’ end of the methylation specific primer.

42. The complex of any one of claims 33-41, wherein one of the flanking arms is located on a 5 ’ end of the methylation specific primer.

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